In the late 19th century, the fact that, when serum of an experimental animal to which diphtheria and tetanus of a non-lethal dose were administered was administered to other animals, diphtheria and tetanus can be prevented was found. After the finding, clinical use of the concept of serum therapy, that is, antibody therapy was gradually started. However, the early antibody treatment had very limited practicability due to problems of obtaining a high purity antibody and contamination by blood-borne infectious agents. In order to address such problems in the traditional antibody treatment, a rodent-originated monoclonal antibody of a pure form was produced on a large scale at a relatively low cost according to a hybridoma fusion technique developed in 1975. However, due to several problems and side effects such as a short half-life, an immune response to an anti-mouse antibody, reduced efficacy, and a fatal allergic reaction when a mouse originated monoclonal antibody is administered to a human body, a clinical use thereof was limited.
Due to the advent of a gene recombinant technique, which was a starting point of a biotechnology revolution in the 1980s, a humanized antibody in which a mouse monoclonal antibody is humanized through gene manipulation could be prepared; various immunological side effects caused when the antibody is administered to a patient were minimized; and the foundation of an active clinical use of a therapeutic antibody was established. Meanwhile, fundamental technology with which a complete human monoclonal antibody can be produced with the aid of a phage display technique or a transgenic mouse has been developed since the mid-1990s. Currently, many domestic and international pharmaceutical companies enthusiastically conduct a great deal of research and investment for developing a new drug using an antibody. Today, US Food and Drug Administration (FDA) approved new antibody drugs numbering about 26 which are commercially available worldwide, and 300 or more therapeutic antibodies are in a clinical trial step, which will show the importance of the antibody in the pharmaceutical industry. Meanwhile, preclinical and clinical trial results showing that, when an antibody having target selectivity and a chemotherapeutic agent having no target specificity are co-administered, side effects are suppressed and a therapeutic effect is improved have been recently reported. Therefore, usefulness of the antibody will be further increased in anti-cancer treatment.
Meanwhile, currently, a new antibody drug is being developed mainly for cancer and autoimmune diseases. In particular, a new antibody drug in the form of IgG or an intact antibody does not show a satisfactory therapeutic effect for solid tumors, and a high antibody production cost may be an obstacle to develop the new antibody drug. Therefore, the development of a new antibody drug of a recombinant protein form that has a more improved biological effect than the antibody in the related art has been continuously attempted. One of these is a bispecific antibody in which one antibody can bind to at least two target molecules, on which a research has been started since the mid-1980s to use the antibody, in particular, for anti-cancer treatment.
Natural antibodies (immunoglobulin G (IgG), IgM, IgD, IgE, and IgA) have a form in which two heavy chains having the same amino acid sequence and two light chains having the same sequence are assembled. In this case, formation of a homodimer of the same two heavy chains is induced through an interaction between the final domains (that is, a CH3 domain in IgG, a CH4 domain in IgM, a CH3 domain in IgD, CH2 and CH4 domains in IgE, and a CH3 domain in IgA) of a constant region (Fc, crystallizable fragment) of an antibody. Then, a disulfide bond between hinge regions is induced, and a homodimer between robust heavy chains is formed. Specifically, an assembly of a heavy chain and a light chain in human IgG1 is induced by a disulfide bond between the 5th Cys in a heavy chain hinge region and the 107th Cys in a kappa light chain.
Therefore, a natural monoclonal antibody (mAb) has a characteristic of bivalent binding to one type of an antigen. On the other hand, the bispecific antibody refers to an antibody in a single molecule form that can simultaneously or alternatively bind to two types of antigens. Such a bispecific antibody is known in the related art as a manipulated protein such as a dispecific or multi-specific antibody that can bind to two or more antigens, and can be prepared using cell fusion, chemical bonding, and recombinant DNA techniques.
In the related art, a bispecific antibody was prepared using a quadroma technique in which somatic cell fusion of two different hybridoma cell lines expressing a mouse monoclonal antibody having desired specificity is used (Milstein and Cuello 1983). In this case, two different light chains are randomly paired in quadroma cell lines to yield a maximum of 10 types of various antibodies, and it is very difficult to separate and purify a desired bispecific antibody from this antibody mixture. Accordingly, complex purification processes were necessary to obtain only a desired bispecific antibody since there exist byproducts that form a wrong pair and a production yield decreases (Morrison 2007).
As a method of addressing such problems, a bispecific antibody form in which antigen binding site fragments of a light chain and a heavy chain are connected by various chains and expressed in a single construct was developed. This form includes forms of single chain diabodies, a tandem single chain fragment variable (scFv) antibody and the like (Holliger and Hudson 2005). Also, a bispecific antibody in which additional antigen binding antibody fragments are fused to the N-terminus or C-terminus of a heavy chain or a light chain of an antibody, which has a similar form to Ig, was prepared (Miller, Meng et al. 2003; Lu, Zhang et al. 2004).
However, the bispecific antibody based on such an antibody fragment assembly has problems in that an expression level decreases due to low stability, antibody aggregation is formed, and immunogenicity increases accordingly (Chan and Carter 2010). Also, the bispecific antibody based on only the antibody fragment assembly has no heavy chain constant region (Fc) of the antibody. Therefore, there is a problem in that the following are absent: stability that increases in association with Fc, a long serum half-life depending on an increased size and binding to an Fc receptor (neonatal Fc receptor, FcRn), an advantage of binding site preservation (protein A and protein G) in a purification process, an antibody-dependent cellular cytotoxicity and a complement-dependent cellular cytotoxicity (Chan and Carter 2010).
Therefore, ideally, it is necessary to develop a bispecific antibody having a structure that is very similar to naturally occurring antibodies (IgG, IgM, IgA, IgD, and IgE) and having a minimum sequence deviation therefrom.
In order to resolve the above problem, it was attempted to develop a bispecific antibody using a knob-into-hole technique. In this technique, through gene manipulation, a mutation is induced in a CH3 domain of two different Ig heavy chains, a hole structure is made in a CH3 domain of one Ig heavy chain, a knob structure is made a CH3 domain of the other Ig heavy chain, and two Ig heavy chains are induced to form a heterodimer (U.S. Pat. No. 7,695,936 B2; Korean Laid-open Patent Application No. 10-2010-0087394). In this case, amino acid residues included in a hydrophobic core contributing to formation of the homodimer between human Ig heavy chain CH3 domains are Leu351, Thr366, Leu368, and Tyr407 according to EU numbering of the amino acid number of the antibody chain (Cunningham, Pflumm et al. 1969). In the knob-into-hole technique, it was reported that, with respect to residues positioned at a hydrophobic core in a CH3 domain interface, a hole structure is made in one heavy chain CH3 domain such that hydrophobic amino acid residues having a large side chain are substituted with hydrophobic amino acids having a small side chain (Thr366Ser, Leu368Ala, Tyr407Val), a knob structure is made in the other heavy chain CH3 domain such that hydrophobic amino acid residues having a small side chain are substituted with hydrophobic amino acids having a large side chain (Thr366Trp), and when two mutation pairs, that is, heavy chain constant region mutation pairs in which CH3A (Thr366Ser, Leu368Ala, and Tyr407Val) and CH3B (Thr366Trp) are introduced were co-expressed, formation of the heterodimeric Fc is preferred more than formation of the homodimer heavy chain constant region (Ridgway, Presta et al. 1996). However, in the knob-into-hole technique, it was reported that a yield of formation of the heterodimeric Fc (heterodimer heterodimeric Fc) is about 80% (Ridgway, Presta et al. 1996). In order to promote stabilization of the heterodimer, there is a case where a phage display and a disulfide bridge are introduced for further increasing the interaction (Atwell, Ridgway et al. 1997; Merchant, Zhu et al. 1998, and U.S. Pat. No. 5,731,168A).
As another method in which formation of the heterodimeric Fc (heterodimer heterodimeric Fc) is enhanced, there is an example where a mutation is induced in a charged amino acid in an interface between CH3 domains. Specifically, a CH3 domain in one constant region is induced to have positively charged side chain amino acids, and a CH3 domain in the other constant region is induced to have negatively charged side chain amino acids, thereby inhibiting formation of the homodimer due to electrostatic repulsion, and enhancing formation of the heterodimer due to electrostatic interaction (Gunasekaran, Pentony et al. 2010, and US Laid-open Patent Application No. 2010/0286374 A1). That is, Lys392Asp and Lys409Asp are introduced into one CH3 domain, Glu356Lys and Asp399Lys are introduced into the other CH3 domain, and thus, formation of the heterodimer was induced. A yield of formation of the heterodimeric Fc of the CH3 domain mutation is about 90%.
As still another method in which formation of the heterodimeric Fc (heterodimer heterodimeric Fc) is enhanced, there is an example where a complementary mutation is induced in a region forming a hydrophobic core region of a CH3 interface due to a size difference between hydrophobic amino acid side chains, and thus, formation of the heterodimer is enhanced (Moore, Bautista et al. 2011, and US Laid-open Patent Application No. US 2011/0054151 A1). Specifically, Ser364His and Phe495Ala are introduced into one CH3 domain, Tyr349Thr and Thr394Phe are introduced into the other CH3 domain, and thus, formation of the heterodimer was induced. A yield of formation of the heterodimeric Fc of the CH3 domain mutation is about 80 to 90%.
However, the heterodimeric Fc including the developed CH3 domain variant pair has a lower thermodynamic stability and expression yield than that of a wild type antibody.
Therefore, it is necessary to develop the heterodimeric Fc having a yield of formation of the heterodimer as high as possible, having a thermodynamic stability and expression yield that is similar to or more increased than that of a wild type, but there is still no report satisfying such necessity.